Viscous Blood Research Articles

Association between Carotid Wall Shear Stress-Based Vascular Vector Flow Mapping and Cerebral Small Vessel Disease.

Wall shear stress (WSS) is the frictional force caused by viscous blood flowing along the vessel wall. Decreased WSS is associated with local vascular endothelial dysfunction and atherosclerosis. The vector flow mapping (VFM) technique detects the direction of intracardiac blood flow and WSS on the vessel wall with echocardiography. In this study, we examined carotid WSS by applying the VFM technique to the carotid arteries and evaluated its relationship with cerebral small vessel disease (SVD). This is a single-center, prospective, observational study. We investigated the association between carotid WSS and SVD imaging, and cognitive outcomes in consecutive 113 patients with acute lacunar infarction. Carotid WSS was negatively associated with age (r=-0.376, p＜0.001). Lower WSS was correlated with total SVD scores (ρ=-0.304, p=0.004), especially with enlarged perivascular space (EPVS) in the basal ganglia ＞10 (p＜0.001). The carotid intima-media thickness was not associated with the total SVD score (ρ=-0.183, p=0.052). Moreover, lower WSS was associated with executive dysfunction. EPVS has recently been reported as a marker of early SVD imaging, and executive dysfunction is common in vascular cognitive impairment. These results suggested that decreased carotid WSS based on vascular VFM, which can be measured easily, is associated with imaging and cognitive changes in the early stages of SVD.

Evaluation of A Highly Viscous Blood Sample In Clinical Biochemistry Laboratory, Its Interpretation and Relevance

Evaluation of A Highly Viscous Blood Sample In Clinical Biochemistry Laboratory, Its Interpretation and Relevance

Influence of cardiac function on intermittent hypoxia in rats fed with high-fat diet

A high-fat diet (HFD) accumulates fat in the cardiovascular system, alters the metabolism, and affects cardiac function. Dyslipidemia is associated with the development of sleep apnea syndrome (SAS), which is associated with intermittent hypoxia (IH); however, it is unclear whether SAS affects cardiac function in patients with dyslipidemia. The purpose of this study was to evaluate how IH affects cardiac function in rats fed with a HFD. Male 5-week-old Sprague-Dawley rats of two groups (normal diet (SD) and HFD) were divided into IH-exposed and unexposed groups. Zinc protoporphyrin-9 (ZnPPIX) was administered as a heme oxygenase-1 (HO-1) inhibitor to the SD and IH + HFD groups, and cardiac function and blood viscosity were examined. In the IH + HFD group, echocardiography showed an increased fractional shortening (FS), which peaked on day 4. Western blot analysis revealed an increase in HO-1 after 2 weeks. This peak continued even after the HO-1 inhibitor and ZnPPIX were administered. One cause of increased FS is the stagnation of blood flow due to an increased blood viscosity. To be able to send highly viscous blood to every corner of the body, the heart must contract strongly. Therefore, HO-1 is released by the body as a biological defense reaction. HO-1 has a vasodilatory effect and suppresses hyper constriction. Thus, IH exposure to HFD causes and drives transient hyper constriction, releasing HO-1 as a biological response. This led to dilated blood vessels, after which the FS returned to normal.

Direct separation and enumeration of CTCs in viscous blood based on co-flow microchannel with tunable shear rate: a proof-of-principle study.

Circulating tumor cells (CTCs), which have extremely low density in whole blood, are an important indicator of primary tumor metastasis. Isolation and enumeration of these cells are critical for clinical applications. Separation of CTCs from massive blood cells without labeling and addition of synthetic polymers is challenging. Herein, a novel well-defined co-flow microfluidic device is presented and used to separate CTCs in viscous blood by applying both inertial and viscoelastic forces. Diluted blood without any synthetic polymer and buffer solution were used as viscoelastic fluid and Newtonian fluid, respectively, and they were co-flowed in the designed chip to form a sheath flow. The co-flow system provides the function of particle pre-focusing and creates a tunable shear rate region at the interface to adjust the migration of particles or cells from the sample solution to the buffer solution. Successful separation of CTCs from viscous blood was demonstrated and enumeration was also conducted by image recognition after separation. The statistical results indicated that a recovery rate of cancer cells greater than 87% was obtained using the developed method, which proved that the direct separation of CTCs from diluted blood can be achieved without the addition of any synthetic polymer to prepare viscoelastic fluid. This method holds great promise for the separation of cells in viscous biological fluid without either complicated channel structures or the addition of synthetic polymers.

Dynamics and apparent permeability of the glycocalyx layer: Start-up and pulsating shear experiments in silico

We present a detailed study of the three-dimensional two-way fluid-structure interactions between viscous blood plasma and the hyperelastic solid nanofibers comprising the Endothelial Glycocalyx Layer, along with a simple analytical model. Examining the elasto-hydrodynamics under startup shear and pulsatile flow conditions, we shed light on mesoscopic events close to microvessel walls under physiologic conditions. Our findings impact the efficiency of paracellular and transcellular transport of biological molecules, plasma ions, and viruses. We correlate macroscopic properties like permeability to dimensionless terms, which can expand possible uses to similarly structured hairy networks.

Squeezing viscous blood through narrow pipes, and other problems of high-altitude polycythaemia.

Squeezing viscous blood through narrow pipes, and other problems of high-altitude polycythaemia.

A predictive multiscale model for simulating flow-induced platelet activation: Correlating in silico results with in vitro results

Flow-induced platelet activation prompts complex filopodial formation. Continuum methods fail to capture such molecular-scale mechanisms. A multiscale numerical model was developed to simulate this activation process, where a Dissipative Particle Dynamics (DPD) model of viscous blood flow is interfaced with a Coarse Grained Molecular Dynamics (CGMD) platelet model. Embedded in DPD blood flow, the macroscopic dynamic stresses are interactively transferred to the CGMD model, inducing intra-platelet associated events. The platelets activate by a biomechanical transductive linkage chain and dynamically change their shape in response. The models are fully coupled via a hybrid-potential interface and multiple time-stepping (MTS) schemes for handling the disparity between the spatiotemporal scales. Cumulative hemodynamic stresses that may lead to platelet activation are mapped on the surface membrane and simultaneously transmitted to the cytoplasm and cytoskeleton. Upon activation, the flowing platelets lose their quiescent discoid shape and evolve by forming filopodia.The model predictions were validated by a set of in vitro experiments, Platelets were exposed to various combinations of shear stresses and durations in our programmable hemodynamic shearing device (HSD). Their shape change was measured at multiple time points using scanning electron microscopy (SEM). The CGMD model parameters were fine-tuned by interrogating a parameter space established in these experiments. Segmentation of the SEM imaging streams was conducted by a deep machine learning system. This model can be further employed to simulate shear mediated platelet activation thrombosis initiation and to study the effects of modulating platelet properties to enhance their shear resistance via mechanotransduction pathways.

Hyaluronan and Its Receptors as Regulatory Molecules of the Endothelial Interface.

On the surface of endothelial cells (ECs) lies the glycocalyx, a barrier of polysaccharides that isolates the ECs from the blood. The role of the glycocalyx is dynamic and complex, thanks to not only its structure, but its vast number of components, one being hyaluronan (HA). HA is a critical component of the glycocalyx, having been found to have a wide variety of functions depending on its molecular weight, its modification, and receptor-ligand interactions. As HA and viscous blood are in constant contact, HA can transmit mechanosensory information directly to the cytoskeleton of the ECs. The degradation and synthesis of HA directly alters the permeability of the EC barrier; HA modulation not only alters the physical barrier but also can signal the initiation of other pathways. EC proliferation and angiogenesis are in part regulated by HA fragmentation, HA-dependent receptor binding, and downstream signals. The interaction between the CD44 receptor and HA is a driving force behind leukocyte recruitment, but each class of leukocyte still interacts with HA in unique ways during inflammation. HA regulates a diverse repertoire of EC functions.

Phylogenic Determinants of Cardiovascular Frailty, Focus on Hemodynamics and Arterial Smooth Muscle Cells.

The evolution of the circulatory system from invertebrates to mammals has involved the passage from an open system to a closed in-parallel system via a closed in-series system, accompanying the increasing complexity and efficiency of life's biological functions. The archaic heart enables pulsatile motion waves of hemolymph in invertebrates, and the in-series circulation in fish occurs with only an endothelium, whereas mural smooth muscle cells appear later. The present review focuses on evolution of the circulatory system. In particular, we address how and why this evolution took place from a closed, flowing, longitudinal conductance at low pressure to a flowing, highly pressurized and bifurcating arterial compartment. However, although arterial pressure was the latest acquired hemodynamic variable, the general teleonomy of the evolution of species is the differentiation of individual organ function, supported by specific fueling allowing and favoring partial metabolic autonomy. This was achieved via the establishment of an active contractile tone in resistance arteries, which permitted the regulation of blood supply to specific organ activities via its localized function-dependent inhibition (active vasodilation). The global resistance to viscous blood flow is the peripheral increase in frictional forces caused by the tonic change in arterial and arteriolar radius, which backscatter as systemic arterial blood pressure. Consequently, the arterial pressure gradient from circulating blood to the adventitial interstitium generates the unidirectional outward radial advective conductance of plasma solutes across the wall of conductance arteries. This hemodynamic evolution was accompanied by important changes in arterial wall structure, supported by smooth muscle cell functional plasticity, including contractility, matrix synthesis and proliferation, endocytosis and phagocytosis, etc. These adaptive phenotypic shifts are due to epigenetic regulation, mainly related to mechanotransduction. These paradigms actively participate in cardio-arterial pathologies such as atheroma, valve disease, heart failure, aneurysms, hypertension, and physiological aging.

Spectral noise-to-signal ratio priority method with application for visible and near-infrared analysis of whole blood viscosity.

Whole blood viscosity (WBV) is a group of important clinical indicators of cardio-cerebral vascular diseases. Existing detection methods for WBV are complex, making them inconvenient for large population screening. Blood viscosity is closely related to the deformability and aggregation of erythrocytes, which are associated with haemoglobin. Haemoglobin has obvious near-infrared (NIR) spectral absorption. Scattering occurs when NIR light enters a viscous blood sample, and its scattering degree is correlated with blood viscosity. Based on repeated spectral measurements and spectral similarity, spectral noise-to-signal ratio (NSR) was proposed to quantify the spectral scattering effect in the blood sample. A novel selection method of piecewise-continuous wavelengths, named NSR priority-partial least squares (NSRP-PLS), was proposed and applied for visible-NIR quantitative analysis of WBV with high, medium and low shear rates [WBV(H), WBV(M), WBV(L)]. Modelling was separately performed by gender to allow for systematic gender differences in blood viscosity. For the NIR-predicted and clinically measured values of the three WBV indicators in independent validation, the root mean square errors for prediction (SEP) were 0.498, 0.222 and 0.193 (mPa·s), respectively. And the correlation coefficients (RP) were 0.927, 0.934 and 0.927, respectively. Compared with the three current well-performing methods (MW-PLS, CARS-PLS and SPA-PLS), the proposed NSRP-PLS method achieved better predictive accuracy. Results indicated that visible-NIR spectroscopy combined with the NSRP-PLS method can be used for the quantitative analysis of WBV. The proposed analytical method is rapid, reagent-free and is scientific and meaningful for cardio-cerebral vascular diseases screening in large populations.

Nucleation and Formation of a Primary Clot in Insect Blood

Blood clotting at wound sites is critical for preventing blood loss and invasion by microorganisms in multicellular animals, especially small insects vulnerable to dehydration. The mechanistic reaction of the clot is the first step in providing scaffolding for the formation of new epithelial and cuticular tissue. The clot, therefore, requires special materials properties. We have developed and used nano-rheological magnetic rotational spectroscopy with nanorods to quantitatively study nucleation of cell aggregates that occurs within fractions of a second. Using larvae of Manduca sexta, we discovered that clot nucleation is a two-step process whereby cell aggregation is the time-limiting step followed by rigidification of the aggregate. Clot nucleation and transformation of viscous blood into a visco-elastic aggregate happens in a few minutes, which is hundreds of times faster than wound plugging and scab formation. This discovery sets a time scale for insect clotting phenomena, establishing a materials metric for the kinetics of biochemical reaction cascades. Combined with biochemical and biomolecular studies, these discoveries can help design fast-working thickeners for vertebrate blood, including human blood, based on clotting principles of insect blood.

Variations in pulsatile flow around stenosed microchannel depending on viscosity.

In studying blood flow in the vessels, the characteristics of non-Newtonian fluid are important, considering the role of viscosity in rheology. Stenosis, which is an abnormal narrowing of the vessel, has an influence on flow behavior. Therefore, analysis of blood flow in stenosed vessels is essential. However, most of them exist as simulation outcomes. In this study, non-Newtonian fluid was observed in stenosed microchannels under the pulsatile flow condition. A polydimethylsiloxane channel with 60% stenosis was fabricated by combining an optic fiber and a petri dish, resembling a mold. Three types of samples were prepared by changing the concentrations of xanthan gum, which induces a shear thinning effect (phosphate buffered saline (PBS) solution as the Newtonian fluid and two non-Newtonian fluids mimicking normal blood and highly viscous blood analog). The viscosity of the samples was measured using a Y-shaped microfluidic viscometer. Thereafter, velocity profiles were analyzed under the pulsatile flow condition using the micro-particle image velocimetry (PIV) method. For the Newtonian fluid, the streamline was skewed more to the wall of the channel. The velocity profile of the non-Newtonian fluid was generally blunter than that of the Newtonian fluid. A highly oscillating wall shear stress (WSS) during the pulsatile phase may be attributed to such a bluntness of flow under the same wall shear rate condition with the Newtonian fluid. In addition, a highly viscous flow contributes to the variation in the WSS after passing through the stenosed structures. A similar tendency was observed in simulation results. Such a variation in the WSS was associated with plaque instability or rupture and damage of the tissue layer. These results, related to the influence on the damage to the endothelium or stenotic lesion, may help clinicians understand relevant mechanisms.

Extreme blood-boosting capacity of an Antarctic fish represents an adaptation to life in a sub-zero environment.

Blood doping, the practice of boosting the oxygen carrying capacity of blood, is an illegal strategy used by human athletes to enhance aerobic capacity and athletic performance. Interestingly, the practice of boosting blood oxygen carrying capacity is also naturally prevalent in the animal kingdom via the splenic release of stored erythrocytes. Here, we demonstrate that an Antarctic notothenioid fish, the bald notothen (Pagothenia borchgrevinki), is a master of this practice. Because of the sub-zero environment these fish inhabit, they sequester a large proportion of erythrocytes in the spleen during times of inactivity to reduce the energetic and physiological costs associated with continuously pumping highly viscous blood around the body. However, in response to metabolically demanding situations (i.e. exercise and feeding), these fish contract the spleen to eject stored erythrocytes into circulation, which boosts blood oxygen carrying capacity by up to 207% (cf. exercise-induced increases of ∼40-60% in a range of other vertebrates and ∼5-25% in blood-doping athletes). By evaluating cardiorespiratory differences between splenectomized (unable to release erythrocytes from the spleen) and sham-operated individuals, we demonstrate the metabolic benefits (i.e. aerobic scope increase of 103%) and the cardiovascular trade-offs (i.e. ventral aortic blood pressure and cardiac workload increase of 12% and 30%, respectively) associated with the splenic blood-boosting strategy. In conclusion, this strategy provides bald notothens with an extraordinary facultative aerobic scope that enables an active lifestyle in the extreme Antarctic marine environment, while minimizing the energetic and physiological costs of transporting highly viscous blood during times of reduced energetic demand.

Hydrodynamics in the intact and denervated aorta in experiment

Aim. As neurogenic processes affect the cardiovascular system and change its function throughout the whole life, the study aims to simulate and study the hydrodynamics of blood flow in an intact and denervated aorta.Methods. A pulse wave going through an intact segment of a dog’s thoracic aorta, a denervated autograft, and a donor dog’s thoracic aorta allograft was assessed by using equipment for blood pressure measurement and tensiometry of longitudinal and diametrical deformations of blood vessels at the point of pressure measuring. The experiments were performed under general endotracheal anesthesia. Two series of acute experiments on replacement of the thoracic aorta by an autograft and an allograft were carried out. Each series had eight dogs. The approach was thoracotomy in the IV intercostal space on the left. A required segment was replaced after heparin infusion while using subclavian-femoral bypass and retrograde autoperfusion.Results. It was found out experimentally that the formation and propagation of a normal isolated pulse wave with preservation of a viscous laminar blood flow were impossible without preserved innervation of the aorta. The experiments showed that in an intact aorta, its diameter tends to increase (by 400–500 μm), with simultaneous shortening of the segment (by 250 μm), and this process keeps being ahead of the pressure wave by 0.02–0.04 s. The rate of aortic dilatation is faster than that of systolic pressure elevation. However, in contrast to the intact aorta, the autograft, on the contrary, becomes longer and narrows, as pressure rises.Conclusion. To create optimal hydrodynamics of aortic blood flow, there should be a neuroreflectory system to control each pulse wave, which must determine its solitonic nature and provide antiflatter stabilization of blood flow.Received 16 April 2018. Revised 3 June 2018. Accepted 7 June 2018.Funding: The study did not have sponsorship.Conflict of interest: The authors declare no conflict of interest.

Advection Flows-Enhanced Magnetic Separation for High-Throughput Bacteria Separation from Undiluted Whole Blood.

A major challenge to scale up a microfluidic magnetic separator for extracorporeal blood cleansing applications is to overcome low magnetic drag velocity caused by viscous blood components interfering with magnetophoresis. Therefore, there is an unmet need to develop an effective method to position magnetic particles to the area of augmented magnetic flux density gradients while retaining clinically applicable throughput. Here, a magnetophoretic cell separation device, integrated with slanted ridge-arrays in a microfluidic channel, is reported. The slanted ridges patterned in the microfluidic channels generate spiral flows along the microfluidic channel. The cells bound with magnetic particles follow trajectories of the spiral streamlines and are repeatedly transferred in a transverse direction toward the area adjacent to a ferromagnetic nickel structure, where they are exposed to a highly augmented magnetic force of 7.68 µN that is much greater than the force (0.35 pN) at the side of the channel furthest from the nickel structure. With this approach, 91.68% ± 2.18% of Escherichia coli (E. coli) bound with magnetic nanoparticles are successfully separated from undiluted whole blood at a flow rate of 0.6 mL h-1 in a single microfluidic channel, whereas only 23.98% ± 6.59% of E. coli are depleted in the conventional microfluidic device.

Transcriptional activation of muscle atrophy promotes cardiac muscle remodeling during mammalian hibernation.

Background. Mammalian hibernation in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is characterized by dramatic changes on a physiological and molecular level. During hibernation, mammalian hearts show a propensity to hypertrophy due to the need for increasing contractility to pump colder and more viscous blood. While cardiac hypertrophy is quite often a process characterized by decompensation, the ground squirrel studied is an excellent model of cardiac plasticity and cardioprotection under conditions of hypothermia and ischemia. The forkhead box O (Foxo) family of proteins and myogenin (MyoG) are transcription factors that control protein degradation and muscle atrophy by regulating the expression of the E3 ubiquitin ligases, MAFbx and MuRF1. These ligases are part of the ubiquitin proteasome system by transferring ubiquitin to proteins and targeting these proteins for degradation. Regulation of Foxo1 and 3a occurs through phosphorylation at different residues. The threonine-24 (Thr-24) and serine-319 (Ser-319) residues on Foxo1, and the Thr-32 residue on Foxo3a are phosphorylated by Akt, leading to cytoplasmic localization of Foxo. We propose that the described mechanism contributes to the changes taking place in cardiac muscle throughout hibernation.Methods. Total and phosphorylated protein levels of Foxo1 and Foxo3a, as well as total protein levels of MyoG, MAFbx, and MuRF1, were studied using immunoblotting.Results. Immunoblotting results demonstrated upregulations in Foxo1 and Foxo3a total protein levels (1.3- and 4.5-fold increases relative to euthermic control, for Foxo1 and 3a respectively) during late torpor, and protein levels remained elevated throughout the rest of torpor and at interbout arousal. We also observed decreases in inactive, phosphorylated Foxo1 and 3a proteins during throughout torpor, where levels of p-Foxo1 Ser319 and Thr24, as well as p-Foxo3a Thr32 decreased by at least 45% throughout torpor. MyoG was upregulated only during late torpor by 2.4-fold. Protein levels of MAFbx and MuRF1 increased in late torpor as well as during early arousal by as much as 2.8-fold, and MAFbx levels remained elevated during interbout arousal, whereas MuRF1 levels returned to control levels.Discussion. The present results indicate that upregulation and activation of Foxo1 and 3a, in addition to the increase in MyoG levels at late torpor, may be upregulating the expression of MAFbx and MuRF1. These findings suggest that there is activation of the ubiquitin proteasome system (UPS) as ground squirrels arouse from torpor. Therefore, the signalling pathway involving MyoG, and the E3 ligases MAFbx and MuRF1, plays a significant role in cardiac muscle remodelling during hibernation. These findings provide insights into the regulation of protein degradation and turnover in the cardiac muscle of a hibernator model.

MECHANICS OF BIOLOGICAL BLOOD FLOW ANALYSIS THROUGH CURVED ARTERY WITH STENOSIS

The viscous fluid model is considered in this article for the study of blood flow through an axis-symmetric stenosis with the effect of three distinct types of arteries i.e., diverging tapering arteries, converging tapering arteries and nontapered arteries. The Cauchy–Euler method has been used for the solution to velocity profile, resistance impedance to flow and the pressure gradient. The characteristics of viscous blood flow on velocity profile, impedance resistance to flow and pressure gradient have been discussed by plotting the graphs of various flow parameters and finally it is found that stenosis dominantes the curvature of curved artery.

Lessons from mammalian hibernators: molecular insights into striated muscle plasticity and remodeling.

Striated muscle shows an amazing ability to adapt its structural apparatus based on contractile activity, loading conditions, fuel supply, or environmental factors. Studies with mammalian hibernators have identified a variety of molecular pathways which are strategically regulated and allow animals to endure multiple stresses associated with the hibernating season. Of particular interest is the observation that hibernators show little skeletal muscle atrophy despite the profound metabolic rate depression and mechanical unloading that they experience during long weeks of torpor. Additionally, the cardiac muscle of hibernators must adjust to low temperature and reduced perfusion, while the strength of contraction increases in order to pump cold, viscous blood. Consequently, hibernators hold a wealth of knowledge as it pertains to understanding the natural capacity of myocytes to alter structural, contractile and metabolic properties in response to environmental stimuli. The present review outlines the molecular and biochemical mechanisms which play a role in muscular atrophy, hypertrophy, and remodeling. In this capacity, four main networks are highlighted: (1) antioxidant defenses, (2) the regulation of structural, contractile and metabolic proteins, (3) ubiquitin proteosomal machinery, and (4) macroautophagy pathways. Subsequently, we discuss the role of transcription factors nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Myocyte enhancer factor 2 (MEF2), and Forkhead box (FOXO) and their associated posttranslational modifications as it pertains to regulating each of these networks. Finally, we propose that comparing and contrasting these concepts to data collected from model organisms able to withstand dramatic changes in muscular function without injury will allow researchers to delineate physiological versus pathological responses.

A one-dimensional model of viscous blood flow in an elastic vessel

In this paper we present a one-dimensional model of blood flow in a vessel segment with an elastic wall consisting of several anisotropic layers. The model involves two variables: the radial displacement of the vessel’s wall and the pressure, and consists of two coupled equations of parabolic and hyperbolic type. Numerical simulations on a straight segment of a blood vessel demonstrate that the model can produce realistic flow fields that may appear under normal conditions in healthy blood vessels; as well as flow that could appear during abnormal conditions. In particular we show that weakening of the elastic properties of the wall may provoke a reverse blood flow in the vessel.

EXPRESSION OF NUCLEAR FACTOR OF ACTIVATED T CELLS (NFAT) AND DOWNSTREAM TARGETS IN GROUND SQUIRREL CARDIAC MUSCLE

13-lined ground squirrels undergo remarkable adaptive changes during hibernation where the animals enter deep torpor and allow body temperature (Tb) to fall as low as 0-5°C. While hibernating, reversible cardiac hypertrophy occurs to increase contractility of the heart; this helps the heart to pump colder and more viscous blood, while avoiding heart failure as a result of sustained or chronic hypertrophy. We were interested in studying the role of the nuclear factor of activated T-cell (NFAT) family of transcription factors and their recently discovered sarcolemmal targets, myoferlin and myomaker, with the adaptations taking place in cardiac muscle during hibernation. Whole heart samples were collected at 6 different time points over the torpor-arousal cycle including euthermic control (EC), entrance into torpor (EN), early torpor (ET), late torpor (LT), early arousal (EA), and interbout arousal (IA) back to euthermia; each stage differs in squirrel Tb and respiration rates. Total protein extracts were prepared and immunoblotting was performed using antibodies specific for mammalian NFATc1, c2, c3, c4, myoferlin, and myomaker. Bioinformatic analysis was conducted on the 1500 bp region upstream of the myomaker transcription start site to identify novel NFAT transcription factor binding sites. DNA-binding enzyme-lined immunosorbent assay (ELISA) was performed over the torpor-arousal timecourse to characterize NFAT-binding to the NFAT-binding sequence identified. In cardiac muscle, NFATc1, c2, and c3 increased during torpor whereas NFATc4 protein levels were elevated throughout the torpor-arousal cycle. A spike in myoferlin protein levels was observed during ET (2.68 fold increase compared to control) with a smaller increase during EA (1.69 fold increase). Myomaker also increased significantly in cardiac muscle during ET (1.88 fold increase) and to a much lesser extent at EA. Bioinformatics analysis and cross-referencing using the NFAT consensus binding sequence resulted in the identification of a putative novel NFAT binding site and DNA-binding ELISAs showed that NFATc1 and c3 were able to bind to this site. This study is the first to observe myomaker expression in cardiac tissue and to identify a possible NFAT-binding region within its promoter. Elevated protein levels of NFATc1-4, myoferlin, and myomaker at selected stages of the torpor-arousal cycle appear to be a regulatory mechanism that contributes to reversible cardiac hypertrophy in response to the changing physiological and environmental stresses encountered during hibernation. The identification of novel targets involved in cardiac hypertrophy provides insight into how heart failure resulting from sustained cardiac overload might be prevented.